Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/76—Chemiluminescence; Bioluminescence

Definitions

• Diagnostic assay reagents often include conjugates of antibodies or other small drug molecules with haptens, such as biotin and fluorescein.
• haptens such as biotin and fluorescein.
• large protein molecules e.g., avidin/streptavidin for biotin and anti-FITC for fluorescein
• Biotin is well known in the art for its use as a food supplement; for example, biotin is utilized to promote healthy hair and nail growth and to treat various disease conditions. Given this use, significant biotin levels can be found in biological samples, such as (but not limited to) blood. Since biotin is used in many diagnostic assays (for example, to coat solid supports), high levels of biotin in test samples can interfere with assay signals in any assays where biotinylated assay components are employed.
• biotin assays Because interference is observed in various assay methods at different biotin concentrations, the levels of biotin present in patient samples should be quantitated to determine if the samples are suitable for use in particular assays. However, most currently available biotin assays have narrow ranges (i.e., 0-50 ng/ml) that are not suitable for detecting the wide dynamic range of biotin concentrations that are typically found in actual patient samples (i.e., 0-1500 ng/ml).
• biotinylated assay component is "pre-bound" with the streptavidin-coated solid support during reagent production. Because of the tight binding and slow off-rate between streptavidin and biotin, replacing the already bound biotin from streptavidin by the incoming biotin in a patient sample is not a predominate process.
• the second way is to increase the streptavidin binding sites on the solid support, so that there are extra binding sites available for the sample biotin molecules in addition to the biotinylated assay components.
• the third way is the combination of both of the above.
• US Patent 5,212,063 teaches the use of polymer particles with a biotin binding core and a covering layer of protein, carbohydrate, or co-polymer for the purpose of filtering free biotin, but does not filter biotin conjugated to large molecules. This approach may be effective for some assay formats, but it also introduces particles that may generate extra absorbance that could interfere with assay signals.
• LOCI ® Luminescent Oxygen Channeling Assay
• U.S. Pat. No. 5,340,716 Ullman et al.
• the currently available LOCI ® technology has high sensitivity and uses several reagents.
• the LOCI ® assay requires that two of these reagents (referred to as a "sensibead” and a “chemibead”) be held by other specific binding partner assay reagents in a manner whereby the sensibead and chemibead are in close proximity to one another to achieve a signal.
• the sensibead Upon exposure to light at a certain wavelength, the sensibead releases singlet oxygen, and if the two beads are in close proximity, the singlet oxygen is transferred to the chemibead; this causes a chemical reaction that results in the chemibead giving off light that can be measured at a different wavelength.
• FIG. 1 schematically depicts multiplex assay components utilized in one non limiting embodiment of the present disclosure.
• FIG. 2 schematically depicts complexes formed from the multiplex assay components of FIG. 1, depending on whether or not biotin and a target analyte are present in a test sample.
• FIG. 3 schematically depicts two non-limiting embodiments of acceptor beads that can be utilized in accordance with the present disclosure (upper panel), and a multiplex assay performed in accordance with the present disclosure (lower panel).
• FIG. 4 depicts an assay sequence for one non-limiting embodiment of the multiplex assay constructed in accordance with the present disclosure, in which two analytes, Protein X (PrX) and biotin are to be measured.
• S sample.
• S biotin sample biotin.
• Abl first antibody to PrX.
• CB chemibead.
• SB sensibead. represents binding.
• FIG. 5 graphically depicts how biotin signal can be translated into a concentration ([biotin] ng/ml) by proper calibration.
• FIG. 6 graphically depicts various scenarios related to biotin interference with a target analyte signal and the correction thereof in one non-limiting embodiment of the multiplex assay according to the present disclosure.
• FIG. 7 contains a table providing a hypothetical scenario of data obtained by the multiplex assay according to the present disclosure and demonstrating the percent interference by sample biotin on target analyte signal.
• FIG. 8 contains one non-limiting method by which target analyte signal can be corrected based upon sample biotin signal or concentration.
• FIG. 9 contains another non-limiting method by which target analyte signal can be corrected based upon sample biotin signal or concentration.
• the use of the term "at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc.
• the term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results.
• the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.
• ordinal number terminology i.e., “first,” “second,” “third,” “fourth,” etc. is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.
• any reference to "one embodiment,” “an embodiment,” “some embodiments,” “one example,” “for example,” or “an example” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment.
• the appearance of the phrase “in some embodiments” or “one example” in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.
• the term "about” is used to indicate that a value includes the inherent variation of error for a composition/apparatus/ device, the method being employed to determine the value, or the variation that exists among the study subjects.
• the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.
• the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
• the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree.
• the term “substantially” means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time.
• the term “substantially adjacent” may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.
• association with and “coupled to” include both direct association/binding of two moieties to one another as well as indirect association/binding of two moieties to one another.
• associations/couplings include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example.
• analog and “derivative” are used herein interchangeably and refer to a substance which comprises the same basic carbon skeleton and carbon functionality in its structure as a given compound, but can also contain one or more substitutions thereto.
• substitution as used herein will be understood to refer to the replacement of at least one substituent on a compound with a residue R.
• R may include H, hydroxyl, thiol, a halogenid selected from fluoride, chloride, bromide, or iodide, a C1-C4 compound selected one of the following: linear, branched or cyclic alkyl, optionally substituted, and linear branched or cyclic alkenyl, wherein the optional substitutents are selected from one or more a I kenyla Ikyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocyclealkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, each of which is optionally substituted wherein the optional substitutents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cyclalkyl, cyclal a
• sample as used herein will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure.
• fluidic biological samples include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.
• binding partner as used in particular (but not by way of limitation) herein in the terms “biotin-specific binding partner” or “target analyte-specific binding partner,” will be understood to refer to any molecule capable of specifically associating with biotin or the target analyte, respectively.
• the binding partner may be an antibody, a receptor, a ligand, aptamers, molecular imprinted polymers (i.e., inorganic matrices), combinations or derivatives thereof, as well as any other molecules capable of specific binding to biotin or the target analyte, respectively.
• antibody is used herein in the broadest sense and refers to, for example, intact monoclonal antibodies and polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the desired biological activity of analyte binding (such as, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody substitute proteins or peptides (i.e., engineered binding proteins/peptides), and combinations or derivatives thereof.
• analyte binding such as, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody
• antibody substitute proteins or peptides i.e.
• the antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2).
• type or class e.g., IgG, IgE, IgM, IgD, and IgA
• sub-class e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2
• hapten refers to a small proteinaceous or non-protein antigenic determinant (or “epitope") which is capable of being recognized by a target analyte-specific binding partner, such as (but not limited to) an antibody.
• a target analyte-specific binding partner such as (but not limited to) an antibody.
• polyhapten as used herein will be understood to refer to a synthetic molecule that contains multiple epitopes/antigenic determinants attached thereto.
• an "analyte” is a macromolecule that is capable of being recognized by an analyte-specific binding partner, such as (but not limited to) an antibody. Both analytes and haptens comprise at least one antigenic determinant or "epitope," which is the region of the antigen or hapten which binds to the analyte-specific binding partner (i.e., antibody). Typically, the epitope on a hapten is the entire molecule.
• signal producing system (sps) members comprise a sensitizer such as, for example, a photosensitizer, and two or more chemiluminescent-fluorescent molecule compositions (wherein a first chemiluminescent composition generates a signal related to the presence of biotin, whereas at least a second chemiluminescent composition generates a signal related to the presence of a target analyte); in these assay embodiments, activation of the sensitizer results in a product that activates the chemiluminescent composition(s), thereby generating a detectable signal that relates to the amount of bound target analyte and bound biotin being detected.
• a sensitizer such as, for example, a photosensitizer
• two or more chemiluminescent-fluorescent molecule compositions wherein a first chemiluminescent composition generates a signal related to the presence of biotin, whereas at least a second chemiluminescent composition generates a
• LOCI ® Luminescence Oxygen Channeling Assay
• target analytes capable of detection via the assay formats described or otherwise contemplated herein may be detected by the multiplex assays of the present disclosure.
• target analytes include: Procalcitonin (PCT), BNP, NT- proBNP, D-Dimer, CKMB, Myoglobin, Myeloperoxidase, ST2, hCG, LH, FSH, iPTH, TSH, fT 4, T 4, PSA, fPSA, and cPSA, and combinations thereof.
• kits for performing multiplex assays that utilize a chemiluminescent detection system for determining the concentration of biotin and at least one additional target analyte in a sample.
• the kit includes: (a) a composition comprising a singlet oxygen-activatable chemiluminescent compound having biotin or an analog thereof directly or indirectly bound thereto, as well as a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a composition comprising a singlet oxygen-activatable chemiluminescent compound having a first analyte-specific binding partner for a target analyte directly or indirectly bound thereto, as well as a fluorescent molecule that is excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecule of (a) and emits light at a different wavelength than the fluorescent molecule of (a); (c) a biotinylated second analyte-specific binding partner for the target analyte, wherein the second analyte-specific binding partner binds to a different epitope of the target analyte than the first analyte-specific binding partner of (b);
• the kit further includes (e) a composition comprising a singlet oxygen-activatable chemiluminescent compound having a first analyte- specific binding partner for a second target analyte directly or indirectly bound thereto, as well as a fluorescent molecule that is excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) a biotinylated second analyte-specific binding partner for the second target analyte, wherein the second analyte-specific binding partner binds to a different epitope of the second target analyte than the first analyte-specific binding partner.
• a composition comprising a singlet oxygen-activatable chemiluminescent compound having a first analyte- specific binding partner for a second target analyte directly or indirectly bound thereto, as well
• a chemiluminescent compound is a compound that is chemically activatable and, as a result of such activation, emits light at a certain wavelength.
• chemiluminescers include: olefins capable of reacting with singlet oxygen or a peroxide to form hydroperoxides or dioxetanes, which can decompose to ketones or carboxylic acid derivatives; stable dioxetanes which can decompose by the action of light; acetylenes which can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azo carbonyls such as (but not limited to) luminol; and aromatic compounds that can form endoperoxides, for example.
• the chemiluminescers directly or indirectly cause the emission of light.
• the singlet oxygen-activatable chemiluminescent compound may be a substance that undergoes a chemical reaction with singlet oxygen to form a metastabile intermediate species that can decompose with the simultaneous or subsequent emission of light.
• the composition comprising the chemiluminescent compound may be directly excited by the activated chemiluminescent compound; alternatively, the composition may further comprise at least one fluorescent molecule that is excited by the activated chemiluminescent compound.
• a sensitizer is a molecule, usually a compound, that generates a reactive intermediate such as, for example, singlet oxygen, for activation of a chemiluminescent compound.
• the sensitizer is a photosensitizer.
• Other sensitizers that can be chemi-activated include, by way of example and not limitation, other substances and compositions that can produce singlet oxygen with or without activation by an external light source. For example, certain compounds have been shown to catalyze the conversion of hydrogen peroxide to singlet oxygen and water.
• Non-limiting examples of other sensitizer substances and compositions include: oxides of the alkaline earth metals Ca, Sr, and Ba; derivatives of elements of groups 3A, 4A, SA, and 6A in d° configuration; oxides of actinides and lanthanides; and oxidizers CIO , BrO , Au 3+ , IC>3 , and I0 4 ; and in particular, molybdate, peroxomolybdate, tungstate, and peroxotungstate ions, and acetonitrile.
• photosensitizers are compounds that are not true sensitizers but which on excitation by heat, light, ionizing radiation, or chemical activation will release a molecule of singlet oxygen.
• Members of this class of compounds include, for example (but not by way of limitation), the endoperoxides such as 1,4- biscarboxyethyl-1, 4-naphthalene endoperoxide; 9,10-diphenylanthracene-9,10- endoperoxide; and 5,6,11,12-tetraphenyl naphthalene 5,12-endoperoxide. Fleating or direct absorption of light by these compounds releases singlet oxygen.
• a photosensitizer is a sensitizer for activation of a photoactive compound, for example, by generation of singlet oxygen by excitation with light.
• the photosensitizers are photoactivatable and include, e.g., dyes and aromatic compounds, and are usually compounds comprised of covalently bonded atoms, usually with multiple conjugated double or triple bonds.
• the compounds should absorb light in the wavelength range of from about 200 nm to about 1,100 nm, such as (but not limited to) a range of from about 300 nm to about 1,000 nm or a range of from about 450 nm to 950 nm, with an extinction coefficient at its absorbance maximum greater than 500 M 1 cm , or greater than 5,000 M 1 cm 4 , or greater than 50,000 M 1 cm 4 , at the excitation wavelength.
• Photosensitizers should be relatively photostable and may not react efficiently with singlet oxygen.
• photosensitizers examples include: acetone; benzophenone; 9-thioxanthone; eosin; 9,10-dibromoanthracene; methylene blue; metallo-porphyrins such as (but not limited to) hematoporphyrin; phthalocyanines; chlorophylls; rose bengal; and buckminsterfullerene; as well as derivatives of these compounds.
• chemiluminescent compounds and photosensitizers that may be utilized in accordance with the present disclosure are set forth in U.S. Pat. No. 5,340,716 (Ullman, et al.), the entire contents of which are hereby expressly incorporated herein by reference.
• any target analyte-specific binding partners known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure.
• analyte-specific binding partners for target analytes include an antibody, a receptor, a ligand, an aptamer, a molecular imprinted polymer (i.e., inorganic matrix), and any combinations or derivatives thereof, as well as any other molecules capable of specific binding to the target analyte.
• each of the first and second analyte-specific binding partners of (a) and (b) is an antibody against the target analyte.
• each of the first and second analyte-specific binding partners of (e) and (f), if present may be an antibody against the second target analyte, in a particular (but non-limiting) embodiment.
• biotin-specific binding partners known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure.
• the biotin-specific binding partner is an antibody against biotin.
• the biotin-specific binding partner is avidin or an analog thereof.
• any avidin analogs known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure, so long as the avidin or avidin analog is: (1) capable of association with the sensitizer; (2) capable of binding to the biotinylated analyte-specific binding partner; and (3) capable of binding to biotin that may be present in a sample.
• avidin analogs that can be utilized in accordance with the present disclosure include those disclosed in Kang et al. (J Drug Target (1995) 3:159-65), the entire contents of which are expressly incorporated herein by reference.
• avidin analogs include avidin, streptavidin, traptavidin, neutral avidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin, other forms of modified or genetically engineered) avidin, esters, salts, and/or derivatives of any of the above, and the like.
• the singlet oxygen-activatable chemiluminescent compound of (a) and/or (b) has a hapten directly or indirectly bound thereto.
• any fluorescent molecules known in the art that are capable of being excited by the activated chemiluminescent compound and emitting light at a particular, detectable wavelength can be utilized in accordance with the present disclosure as the fluorescent molecules of (a) and (b) (as well as (e), if present), so long as the signals produced by each fluorescent molecule is detectable from the signals produced by the other fluorescent molecules utilized. That is, the fluorescent molecule of (a) must emit light at a wavelength that is sufficiently different from the wavelength at which the fluorescent molecule of (b) emits light so that the two signals can be distinguished from one another when detected simultaneously.
• each fluorescent molecule utilized in accordance with the present disclosure is independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosium.
• terbium emits light at a wavelength of about 545 nm
• uranium emits light at a wavelength of about 612 nm
• samarium emits light at a wavelength of about 645 nm.
• the assay components/reagents of the compositions/kits/microfluidic devices/methods may be provided in any form that allows them to function in accordance with the present disclosure.
• each of the reagents may be provided in liquid form and disposed in bulk and/or single aliquot form within the kit.
• one or more of the reagents may be disposed in the kit in the form of a single aliquot lyophilized reagent.
• the use of dried reagents in microfluidics devices is described in detail in US Patent No. 9,244,085 (Samproni), the entire contents of which are hereby expressly incorporated herein by reference.
• kits may further contain other reagent(s) for conducting any of the particular assays described or otherwise contemplated herein.
• additional reagent(s) will depend upon the particular assay format, and identification thereof is well within the skill of one of ordinary skill in the art; therefore, no further description thereof is deemed necessary.
• the components/reagents present in the kits may each be in separate containers/compartments, or various components/reagents can be combined in one or more containers/compartments, depending on the cross-reactivity and stability of the components/reagents.
• the kit may include a microfluidics device in which the components/reagents are disposed.
• kits can vary widely to provide for concentrations of the components/reagents that substantially optimize the reactions that need to occur during the assay methods and further to optimize substantially the sensitivity of an assay.
• one or more of the components/reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit may further include excipient(s) for dissolution of the dried reagents; in this manner, a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present disclosure can be obtained from these components.
• Positive and/or negative controls may also be included with the kit.
• the kit can further include a set of written instructions explaining how to use the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein.
• Certain additional non-limiting embodiments of the present disclosure are directed to a microfluidics device that includes the components of any of the kits described herein above.
• certain non-limiting embodiments include a microfluidics device for determining the concentration of biotin and at least one additional target analyte in a sample.
• the microfluidics device comprises (i) an inlet channel through which a sample is applied; and (ii) at least a first compartment capable of being in fluidic communication with the inlet channel.
• the compartment(s) of (ii) contains: (a) at least a first composition comprising a singlet oxygen-activatable chemiluminescent compound and having biotin or an analog thereof directly or indirectly bound thereto, the first composition further comprising a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) at least a second composition comprising a singlet oxygen-activatable chemiluminescent compound and having a first analyte-specific binding partner for a target analyte directly or indirectly bound thereto, the second composition further comprising a fluorescent molecule that is excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecule of (a) and emits light at a different wavelength than the fluorescent molecule of (a); (c) a biotinylated second analyte-specific binding partner for the target analyte, wherein the second analyte-specific binding partner binds to a different epitope of the
• the compartment(s) of (ii) further contains at least a third composition comprising a singlet oxygen-activatable chemiluminescent compound and having a second target analyte or an analog thereof directly or indirectly bound thereto, the second composition further comprising a fluorescent molecule that is excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b).
• any of the singlet oxygen-activatable chemiluminescent compounds, sensitizers, fluorescent molecules, biotin or analogs thereof, target analytes or analogs thereof, and target analyte-specific binding partners described in detail herein above or otherwise contemplated herein may be utilized in the microfluidics devices of the present disclosure.
• the singlet oxygen-activatable chemiluminescent compounds of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastabile intermediate species that can decompose with the simultaneous or subsequent emission of light.
• the sensitizer is a photosensitizer.
• each of the first and second analyte-specific binding partners of (b) and (c) is an antibody against the target analyte.
• the biotin-specific binding partner of (iii) is avidin or an analog thereof, or is an antibody against biotin.
• the singlet oxygen-activatable chemiluminescent compound of (a) and/or (b) has a hapten directly or indirectly bound thereto.
• the fluorescent molecules of (a) and (b) are each independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosium.
• terbium emits light at a wavelength of about 545 nm
• uranium emits light at a wavelength of about 612 nm
• samarium emits light at a wavelength of about 645 nm.
• the compartment(s) of (ii) further contains: (e) at least a third composition comprising a singlet oxygen-activatable chemiluminescent compound and having a first analyte-specific binding partner for a second target analyte directly or indirectly bound thereto, the second composition further comprising a fluorescent molecule that is excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) a biotinylated second analyte-specific binding partner for the second target analyte, wherein the second analyte-specific binding partner binds to a different epitope of the second target analyte than the first analyte-specific binding partner of (e).
• the fluorescent molecule of the third composition is selected
• all of elements (a)-(d) (and (e) and (f), when present) of (ii) are present in the same compartment.
• elements (a)-(d) (and (e) and (f), when present) are split between two or more compartments.
• the device may be provided with any arrangement of the compartments and distribution of the various components therebetween that allows the device to function in accordance with the present disclosure.
• any of the compartments of the microfluidics device may be sealed to maintain reagent(s) disposed therein in a substantially air tight environment until use thereof; for example, compartments containing lyophilized reagent(s) may be sealed to prevent any unintentional reconstitution of the reagent.
• the inlet channel and a compartment, as well as two compartments, may be described as being "capable of being in fluidic communication" with one another; this phrase indicates that each of the compartment(s) may still be sealed, but that the two compartments are capable of having fluid flow therebetween upon puncture of a seal formed therein or therebetween.
• microfluidics devices of the present disclosure may be provided with any other desired features known in the art or otherwise contemplated herein.
• the microfluidics devices of the present disclosure may further include a read chamber; the read chamber may be any of the compartments containing the reagents described herein above, or the read chamber may be in fluidic communication with said compartment.
• the microfluidics device may further include one or more additional compartments containing other solutions, such as (but not limited to) wash solutions, dilution solutions, excipients, interference solutions, positive controls, negative controls, quality controls, and the like. These additional compartment(s) may be in fluidic communication with one or more of the other compartments.
• the microfluidics device may further include one or more compartments containing a wash solution, and these compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device.
• the microfluidics device may further include one or more compartments containing an excipient for dissolution of one or more dried reagents, and the compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device.
• the microfluidics device may include one or more compartments containing a dilution solution, and the compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device.
• any of the kits/microfluidics devices described or otherwise contemplated herein may include more than one target analyte assay multiplexed with the biotin assay in a single kit/device.
• each of the assays may be constructed and function as described herein.
• the biotin and target analyte assays described herein may be multiplexed with any other target analyte assay known in the art that is capable of being contained within the kits/microfluidics devices of the present disclosure.
• Non-limiting examples of other assays that may be multiplexed with the assays disclosed and claimed herein include BNP, NT- proBNP, D-Dimer, CKMB, Myoglobin, Myeloperoxidase, ST2, PCT, hCG, LH, FSH, iPTH, TSH, fT 4, T 4, PSA, fPSA, and cPSA, and combinations thereof.
• multiple inlet channels may be connected to the sample application chamber.
• a portion of the sample may be passed from the sample application chamber to the multiple inlet channels without regard for the content thereof.
• structure(s) may be present in the sample application chamber, the inlet channels, and/or the connection therebetween that allow for separation of certain components from the whole sample and delivery of said components to the different assays.
• Certain non-limiting embodiments are also directed to a method for detecting the presence and/or concentration of biotin and at least one additional target analyte in a sample.
• the method comprises the following steps.
• a sample suspected of containing biotin and at least one additional target analyte is combined, either simultaneously or wholly or partially sequentially, with: (a) at least a first composition comprising a singlet oxygen-activatable chemiluminescent compound and having biotin or an analog thereof directly or indirectly bound thereto, the first composition further comprising a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) at least a second composition comprising a singlet oxygen-activatable chemiluminescent compound and having a first analyte-specific binding partner of a target analyte directly or indirectly bound thereto, the second composition further comprising a fluorescent molecule that is excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecule of (a) and emits light at a different wavelength than the fluorescent molecule of (a); (c) a biotinylated second analyte-specific binding partner for the
• the components are incubated together to allow for the binding of (b) and (c) to target analyte present in the sample as well as the binding of (c) to (d), wherein the indirect binding of (b) to (d) (via (c) and target analyte) results in the formation of a target analyte complex in which the sensitizer is brought into close proximity to the chemiluminescent compound.
• (a) binds to (d) in the absence of biotin in the sample and results in the formation of a biotin complex in which the sensitizer is brought into close proximity to the chemiluminescent compound.
• the sensitizer is activated to generate singlet oxygen, wherein activation of the sensitizers present in the biotin complex and in the target analyte complex causes the activation of the chemiluminescent compound present in each complex.
• the amount of chemiluminescence generated by the activated chemiluminescent compound in the biotin complex is determined by measuring the amount of light emitted by the fluorescent molecule of (a), wherein the amount of biotin in the sample is inversely proportional to the amount of light emitted.
• the amount of chemiluminescence generated by the activated chemiluminescent compound in the target analyte complex is determined by measuring the amount of light emitted by the fluorescent molecule of (b) to determine the amount of target analyte in the sample.
• the second - fifth steps can be optionally repeated, if desired.
• step (5) the result of step (5) is corrected based upon any biotin interference detected in step (4).
• the result of step (5) is flagged as unreliable if the biotin concentration detected in step (4) is above a maximum threshold level.
• step (1) further comprises combining with elements (a)-(d): (e) at least a third composition comprising a singlet oxygen-activatable chemiluminescent compound having a first analyte-specific binding partner for a second target analyte directly or indirectly bound thereto, the third composition further comprising a fluorescent molecule that is excited by the activated chemiluminescent compound, wherein the fluorescent molecule is different from the fluorescent molecules of (a) and (b) and emits light at a different wavelength than the fluorescent molecules of (a) and (b); and (f) a biotinylated second analyte-specific binding partner for the second target analyte, wherein the second analyte-specific binding partner binds to a different epitope of the second target analyte than the first analyte-specific binding partner of (e).
• a third composition comprising a singlet oxygen-activatable chemiluminescent compound having a first analyte-
• the method may further comprise the steps of: (8) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the second target analyte complex by measuring the amount of light emitted by the fluorescent molecule of (e) to determine the amount of target analyte in the sample; and (9) correcting the result of step (8) based upon any biotin interference detected in step (4) and/or flagging the result of step (8) as being unreliable if the biotin concentration detected in step (4) is above a maximum threshold level.
• any of the singlet oxygen-activatable chemiluminescent compounds, sensitizers, fluorescent molecules, biotin or analogs thereof, target analytes or analogs thereof, and target analyte-specific binding partners described in detail herein above or otherwise contemplated herein may be utilized in the methods of the present disclosure.
• the singlet oxygen-activatable chemiluminescent compounds of (a) and (b) (and (e), when present) are substances that undergo a chemical reaction with singlet oxygen to form a metastabile intermediate species that can decompose with the simultaneous or subsequent emission of light.
• the sensitizer is a photosensitizer
• the activation of the sensitizer in step (3) comprises irradiation with light (such as, but not limited to, irradiation at about 680 nm).
• each of the first and second analyte-specific binding partners of (b) and (c) (as well as (e) and (f), if present) is an antibody against the target analyte.
• the biotin-specific binding partner of (d) is avidin or an analog thereof, or is an antibody against biotin.
• the singlet oxygen-activatable chemiluminescent compound of (a) and/or (b) has a hapten directly or indirectly bound thereto.
• the fluorescent molecules of (a) and (b) (and (e), if present) are each independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosiu m.
• terbium emits light at a wavelength of about 545 nm
• uranium emits light at a wavelength of about 612 nm
• samarium emits light at a wavelength of about 645 nm.
• samples include a biological sample such as, but not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.
• a biological sample such as, but not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.
• CSF cerebrospinal fluid
• Particular non-limiting examples include lysed whole blood cells and lysed red blood
• the various components of the method are provided in combination (either simultaneously or sequentially).
• the order of addition of the components may be varied; a person having ordinary skill in the art can determine the particular desired order of addition of the different components to the assay.
• the simplest order of addition is to add all the materials simultaneously and determine the signals produced therefrom.
• each of the components, or groups of components can be combined sequentially.
• an incubation step may be involved subsequent to one or more additions.
• step (1) of the method includes first combining the sample with the biotinylated target analyte-specific binding partner and the composition comprising the sensitizer and incubating same before adding the compositions comprising the singlet oxygen-activatable chemiluminescent compounds.
• step (1) of the method can include first combining the sample with the compositions comprising the singlet oxygen-activatable chemiluminescent compounds and incubating same before adding the composition comprising the sensitizer.
• the biotinylated target analyte-specific binding partner may be added before or after the incubation step.
• the present disclosure is also directed to other assay formats (and kits, microfluidics devices, and methods of performing same) for which elimination of sample biotin interference is desired.
• the present disclosure also includes assay formats where different signal molecules such as (but not limited to) different antibodies linked to different enzymes that generate signals at different wavelengths can be utilized in place of the chemiluminescent compound-containing compositions described herein above.
• FIG. 1 depicts the assay components present in the kits and microfluidic devices and used in the methods of certain non-limiting embodiments of the present disclosure. These components include (from left to right):
• a first chemibead having biotin or an analog thereof directly or indirectly bound thereto and containing a first fluorescent molecule that is excited by activated chemiluminescent compound present in the chemibead;
• a second chemibead having target analyte or an analog thereof directly or indirectly bound thereto and containing a second fluorescent molecule that is excited by activated chemiluminescent compound present in the chemibead.
• the first and second fluorescent molecules are different from one another and emit light at different wavelengths; in this manner, assays for both biotin and target analyte can be performed simultaneously in the same reaction, as complexes containing (i) are detected at a different wavelength from complexes containing (iv).
• biotin When biotin is not present in the sample, a complex is formed between (i) and (ii), as shown on the left side of the upper panel of FIG. 2, and a signal is generated upon activation of the sensitizer. However, when biotin is present in the sample, it competes with (i) for binding to (ii), as shown in the left side of the lower panel of FIG. 2. Thus, the presence of biotin in the sample is detected as a decrease in the signal detected at the first wavelength used to detect light emitted from the first fluorescent molecule.
• the result obtained from the target analyte assay con be corrected based upon any biotin interference detected using the first chemibead and/or flagged as unreliable if the detected biotin concentration is above a maximum threshold level.
• the multiplex assays of the present disclosure can be adapted to simultaneously detect more than one target analyte in addition to the detection of biotin interference.
• additional components like (iii) and (iv) are added, wherein the fluorescent molecule present in the additional component like (iv) differs from the first and second fluorescent molecules in that it emits light at a different and separately detectable wavelength than the first and second fluorescent molecules.
• two, three, four, five, six, seven, eight, nine, ten, or more target analytes can be detected in a single reaction, so long as the chemibeads used to detect each contain different fluorescent molecules that each emit light at different and separately detectable wavelengths; as such, the limiting factor in how many target analytes can be detected in a single reaction are the number of fluorescent molecules available that function as described herein.
• This Example provides a multiplexed assay for biotin and procalcitonin (PCT).
• Biotin is covalently conjugated to chemibeads dyed with terbium (FIG. 3, left side of upper panel), and capture antibody for PCT is covalently conjugated to chemibeads dyed with europium (FIG. 3, right side of upper panel).
• the multiplexed method is calibrated with a multi-analyte calibrator containing at least both biotin and PCT. The same streptavidin sensibeads are used for detecting both biotin and PCT.
• the sensibeads are activated by excitation light at 680 nm to generate singlet oxygen, and singlet oxygen diffuses into both types of chemibeads.
• the biotin chemibeads When complexed with biotin, the biotin chemibeads emit light at 545 nm, which is measured as a biotin signal.
• the PCT chemibead When complexed with PCT, the PCT chemibead emit light at 612 nm, which is measured as a PCT signal.
• Two calibration curves are then generated from the multiplex measurements: one for biotin and one for PCT.
• the measured biotin concentration in a sample using terbium as the fluorescent molecule is related to the biotin LOCI ® signal measured using europium as the fluorescent molecule.
• the LOCI ® signal at varying biotin concentrations measured by europium is experimentally obtained.
• the biotin LOCI ® signal by europium at certain biotin concetration can then be predicted (extrapolated) and, if necessary, subsequently subtracted (if a positive impact on signal is observed) or added (if a negative impact on signal is observed) to correct the results of the target analyte.
• the target analyte assay results can be corrected using the biotin signal deduced from the biotin measurement in multiplex fashion.
• the result can be flagged as unreliable if the detected biotin concentration is above a maximum threshold level.
• FIG. 4 depicts an assay sequence for one non-limiting embodiment of the multiplex assay constructed in accordance with the present disclosure, in which two analytes are to be measured: the target analyte (referred to in this Example as Protein X (PrX)) and biotin.
• the assay for the target analyte PrX is a sandwich assay that requires the use of two antibodies; the first antibody (Abl) is coated on chemibeads (Abl-CBs), and the second antibody (Ab2) is biotinylated.
• the biotin assay uses a competitive format and requires the use of biotin- (or biotin analog-) coated chemibeads (Biotin-CBs).
• FIG. 4 depicts one addition sequence for this multiplex assay, such sequence is not to be considered limiting to the present disclosure; indeed, the various assay components may be added in any order, and the two assays may be performed in any desired sequence. Thus, the order of addition and sequence of assays shown in FIG. 4 is non-limiting to the scope of the present disclosure.
• a biotin signal obtained from the biotin assay described above can be translated into a sample biotin concentration ([biotin] ng/ml) by proper calibration utilizing a standard curve, as shown in FIG. 5 (and described in detail in US Serial No. 62/711,694 (filed July 30, 2018). In certain non-limiting embodiments, this concentration may be obtained prior to assaying for the target analyte.
• the calculated biotin concentration may be utilized in one of four different ways:
• biotin signal or calculated biotin concentration is below a threshold above which it is known that biotin starts interfering with the target analyte assay, then report the target analyte assay result as normal.
• biotin signal or calculated biotin concentration is above a threshold level (i.e., above the level at which it is known that biotin starts interfering with the target analyte assay), then the target analyte result is flagged as unreliable and is not reported.
• biotin signal or calculated biotin concentration is above the threshold level, utilize the biotin concentration or signal value to correct the target analyte assay result.
• This option utilizes a combination of (1), (2), and (3) - i.e., correct the affected target analyte assay result when the biotin level is above the threshold level for affecting the target analyte assay but not overwhelmingly exceeding the threshold level. If the biotin signal/calculated concentration detected is low (i.e., below a threshold level, such that biotin does not interfere with the target analyte results), the target analyte result is reported as normal. If the biotin signal/calculated concentration detected is too high (i.e., above a maximum threshold level) and correction is not possible, then a flag should be tripped, and the target analyte result flagged as unreliable.
• FIG. 6 graphically depicts these various scenarios related to biotin interference with a target analyte signal and the possible correction thereof.
• the uppermost curve indicates the assay values obtained when no biotin is present.
• a low biotin concentration is present that has a minimal impact on the target analyte signal; in this scenario, no correction of the target analyte result is needed.
• the biotin concentration/signal exceeds a threshold value above which biotin interferes with the target analyte assay; in this scenario, correction of the target analyte result based upon the amount of biotin interference detected is required.
• the biotin concentration or signal value is utilized to correct the target analyte assay result.
• the biotin concentration or signal value is significantly above the threshold value and exceeds a maximum threshold value above which the target analyte concentration can no longer be corrected; thus, the amount of biotin interference present causes a flag to be tripped to indicate that the target analyte result is unreliable.
• the upper table in FIG. 7 depicts a hypothetical scenario of target analyte (PrX) signal (in kilocounts) obtained by the multiplex assay according to the present disclosure in the absence and presence of sample biotin.
• PrX target analyte
• the upper table of FIG. 7 depicts the actual target analyte signal detected
• the lower table of FIG. 7 depicts the percent interference by sample biotin on PrX signal (based on the values in the upper table). As can be seen, a different percentage of interference is seen at different PrX concentrations, even when the sample biotin concentration remains constant.
• FIGS. 8 and 9 illustrate two methods by which the target analyte signal can be corrected based upon the sample biotin signal/concentration detected.
• the method illustrated in FIG. 9 utilizes all of the data present in FIG. 7, whereas the method illustrated in FIG. 8 utilizes a subset of the data from FIG. 7.
• FIGS. 8 and 9 illustrate two methods by which the target analyte signal can be corrected based upon sample biotin signal/concentration detected.
• the method illustrated in FIG. 9 utilizes all of the data present in FIG. 7, whereas the method illustrated in FIG. 8 utilizes a subset of the data from FIG. 7.
• the shaded rectangle in the upper table (which is simply reproduced from FIG. 7) is utilized to correct the PrX signal using the sample biotin signal/concentration to provide a more localized correction of the PrX signal.
• the values from the shaded rectangle are rearranged and then entered into statistical software that conducts a regression to obtain a correction equation based on the data from the shaded rectangle.
• the regression correction equation obtained in this example is shown at the bottom of FIG. 8.
• This step can be completed during co-calibration (either in manufacturing or by customer calibration) with calibrators containing both PrX and biotin at varying known concentrations.
• the two-analyte co-calibration creates a surface equation separate from the calibrations for each of biotin and PrX.
• the biotin or PrX single analyte calibration is done in the absence of the other analyte. But this co-calibration is performed when both analytes are present at varying concentrations.
• the purpose of the co-calibration is to obtain a surface equation where PrX concentration (or PrX signal at 0 ng/ml biotin) serves as a function of PrX signal in the presence of biotin interference).
• the reason for using subsets of data in the method of FIG. 8 is to ensure more accurate regression for correction at a more local level. So if the PrX signal and biotin signal/concentration (ng/ml) are known, then an accurate PrX concentration (ng/ml) can be calculated.
• the corrected PrX concentration can be calculated by performing a similar regression that utilizes the entire table or the entire assay ranges (biotin and PrX) obtained, as shown in FIG. 9.
• the data from FIG. 7 has simply been rearranged into the table shown. Then a regression analysis is performed as described herein above to obtain a regression equation, as shown on the right side of FIG. 9.
• FIGS. 8 and 9 both produce regression equations to predict target analyte (PrX) concentration in the presence of biotin interference.
• compositions, kits, and devices as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove.
• present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

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